Project Description: The broad, long-term objective of this research program is to develop general methods to conditionally regulate protein function at the level of the protein molecules rather than by targeting the precursor DNA or mRNA sequences that encode a particular protein of interest. This new technology should be completely specific for the targeted protein and should provide rapid and dose-dependent control of protein function using cell-permeable small molecules. Cells typically regulate protein expression levels by balancing the creation and degradation of protein molecules. The majority of existing perturbation methods such as transcriptional switches and RNAi focus on protein synthesis, however when synthesis is switched off there is inevitably an experimental delay as the existing protein must be degraded. This research proposal takes a fundamentally different approach by focusing on methods to conditionally target specific proteins for degradation. The goal is to engineer small protein domains that are rapidly and constitutively degraded when expressed in mammalian cells. Importantly, the instability of a destabilizing domain is faithfully conferred to other proteins fused to these small domains. Addition of a cell-permeable ligand that binds tightly to the destabilizing domains shields them from degradation, allowing the fused proteins to perform their cellular functions. The genetic fusion of the destabilizing domain to the gene-of-interest ensures specificity, and the attendant small-molecule control confers speed, reversibility and dose-dependence to this method. The specific aims of this proposal focus on developing new destabilizing domains that provide conditional control of protein expression to membrane-bound proteins expressed in mammalian cells as well as essential proteins expressed in yeast. This technology will be used to conditionally regulate enzymes involved in epigenetic remodeling of differentiated cells. Relevance: The goal of this research is to develop general new methods to rapidly and reversibly regulate the expression of specific proteins in eukaryotic cells. This methodology would enable the development of many new models for human diseases (including cultured cells or knock-out mice), and these model systems are enormously helpful in the ongoing search for new and improved drugs to treat human diseases.